Methods,  compositions and systems for  production of recombinant spider silk polypeptides

ABSTRACT

Disclosed are methods, compositions, and systems for transforming silkworms to produce spider silk and analogs of spider silk. In certain embodiments, the method may include inserting a DNA sequence coding for at least a portion of a spider silk fibroin polypeptide, or an analog of a spider silk fibroin polypeptide, positioned between at least a portion of the 5′ and 3′ ends of a silkworm fibroin gene to generate a fusion gene construct having a sequence that encodes for a polypeptide comprising both spider silk fibroin and silkworm silk fibroin sequences. In certain embodiments, the fused gene is able to replace a native gene present in the silkworm such that the transformed silkworm expresses a polypeptide comprising a spider silk fibroin polypeptide, or an analog thereof, and expresses significantly less of the native silkworm silk.

PRIORITY CLAIM TO RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No.14/843,363, filed Sep. 2, 2015, which is a division of U.S. applicationSer. No. 12/363,326, filed Jan. 30, 2009, now U.S. Pat. No. 9,131,671,which claims priority under 35 U.S.C. § 119(e) of ProvisionalApplication No. 61/025,616, filed Feb. 1, 2008, and ProvisionalApplication No. 61/037,937, filed Mar. 19, 2008. The entire contents ofeach of these applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods, compositions and systems forproduction of recombinant spider silk polypeptides.

BACKGROUND OF THE INVENTION

Spider silk is a natural fiber with exceptional properties. Draglinesilks in particular possess tensile strength equal to, and a toughnessthat exceeds, KEVLAR™ (Gosline et al., Endeavour, 10, 37-43, (1986);Denny, M. W. J. Exp. Biol., 65, 483-506 (1976); and Lucas, F. Discovery,25, 20-26(1964)). As a silk fiber, spider silk has the texture andflexibility of silk produced by silkworms (e.g., Bombyx mori). Thus,spider silk can be processed, woven, and dyed in the same manner andusing the same equipment used for the processing of silkworm silk.However, spider silk has much more strength and elasticity than silkderived from the silkworm, giving textiles derived from spider silkunique properties. Spider silk can be used as a direct replacement forKEVLAR™, Spectra™, and other high strength fibers giving stronger,lighter, and more flexible products.

Spider silk is composed of large proteins, made up of alternating betasheets and amorphous domains (Lucase, F. et al, J. Text Inst., 46,T440-T452 (1985); Hepburn, H. R. et al. Insect BioChem., 9, 69-77(1979); and Warwicker, J. O., J. Mol. Biol., 2, 350-362 (1960)). Thebeta sheet domains are believed to be responsible for the strength ofsilks. It has been suggested that similar to rubber, the elasticity ofspider silk is entropy driven, and that the amorphous sections betweenthe beta sheets are responsible for much of the elasticity (Gosline etal, Nature, 309, 551-552, (1984); Hepburn, H. R. et al, Insect Biochem.,9, 69-77 (1979)).

The formation of silk from the precursor dope solution is a complexbiological, chemical, and physical process. This complex interaction hasapparently been maximized in arthropods such as spiders and moths, buthas yet to be replicated artificially by humans.

For example, the genes for several spider silks have been identified andcloned. Also, attempts have been made to design peptides that displaysimilar biological and physiological characteristics to spider silk(i.e., “spider silk analogs). Expression of such native spider silkpeptides and potential spider silk analog peptides in bacteria, insectcell lines, goats, and plants has been achieved. However, attempts tospin silk from the purified precursors have not met with success, inpart because the resulting fiber(s) did not properly replicate thequalities of native spider silk.

Spiders are solitary, cannibalistic arthropods and as such, are notparticularly well suited for use as bioreactors. Additionally, spidersonly produce short segments of fiber in limited quantities. Silkworms,on the other hand, produce filaments exceeding 1000 meters in length.Additionally, silkworms produce large quantities of silk; the annualworld production approaches 100 million kilograms. Bombyx mori silkwormshave been used as a bioreactor to produce a number of proteins andpeptides, but the expression systems have generally been found to berelatively unstable and of short duration. Also, the exact mechanismsfor the synthesis, modification, internal transport, and spinning of thesilk fiber in Bombyx mori are not clearly known. Until these mechanismsare elucidated, the use of the natural genetic, cellular, andorganelle/organ systems are most likely to give large quantities of highquality silk fiber.

Production of transgenic silkworms by use of piggyBac transposons isdescribed in U.S. Pat. No. 6,872,869, where a portion of a spider silkgene was fused with a portion of the light chain fibroin of Bombyx underthe control of the promoter of the light chain fibroin. The in-framefusion gene was linked to a reporter gene and then the construct wasligated in between two inverted terminal repeats of the piggybacktransposon. The first plasmid having the fusion gene and insertionsequences was transfected with a second plasmid encoding the transposaseinto silkworm eggs. These insertions produced silk reported to be 30%spider silk mixed with normal Bombyx silk. As the silkworm heavy fibroinchain is approximately 340 kilodaltons (kD), and the fusion protein wasabout 30 kD, a 30% level of spider silk should in fact, correspond to aweight percent of about 5-15%. Thus, because the natural genes are stillpresent and active, the silk produced using these systems includes asignificant amount of the less desirous Bombyx silk. Similar results of10% levels of spider silk in silkworms have been informally reported byanother group (see e.g., Zhang et al, Mol Biol Rep. 2007 May 25;17525867, and the Times (UK) 10 December, 2007).

Thus, there is a significant problem in producing high strength spidersilk, or spider silk analogs, from silkworms. It would be beneficial toproduce silkworms that can generate significant amounts of spider silk,or a spider silk analog, that exhibits the characteristics of spidersilk. The present invention addresses this problem by using the Bombyxsilkworm, which is well-suited as a bioreactor, to produce a silk thatsolely or primarily consists of spider silk, or a spider silk analog,with little or no contamination by the lower strength natural Bombyxsilk.

SUMMARY OF THE INVENTION

Embodiments of the present invention comprise methods, compositions andsystems for transforming a first organism to produce silk polypeptides,or silk polypeptide analogs, from a second organism. For example, incertain embodiments, the present invention comprises methods to generatesilkworms such as Bombyx mori that produce spider silk, and/or analogsof spider silk.

Embodiments of the present invention also comprise recombinant DNAconstructs that provide for the production of such silk. In certainembodiments, the spider silk polypeptide, or spider silk polypeptideanalog, is encoded by a recombinant DNA comprising DNA encoding a spidersilk polypeptide (or analog thereof) fused to 5′ and 3′ DNA sequencesfrom the silkworm silk gene so as to allow the recombinant DNA to insertinto the silkworm silk gene locus by homologous recombination. Thus incertain embodiments of the methods, compositions and systems of thepresent invention, the spider silk gene or analog thereof encoded by therecombinant DNA construct replaces a silkworm gene in genome of thesilkworm.

For example, in one embodiment, the method comprises ligating a DNAsequence coding for at least a portion of a spider silk fibroinpolypeptide, or an analog of a spider silk fibroin polypeptide, betweenat least a portion of the 5′ and 3′ ends of a silkworm fibroin gene togenerate a fused gene having a sequence that encodes for a polypeptidecomprising both spider silk fibroin and silkworm silk fibroin amino acidsequences (i.e., a spider silk/silkworm silk fusion gene). In someembodiments, both the 5′ end of the silkworm fibroin gene and the 3′ endof the silkworm fibroin gene are long enough to allow for homologousrecombination to take place, such that when the genetically modifiedfibroin gene comprising a fused spider/silk silkworm gene is insertedinto a silkworm, it is able to replace the native gene present in thesilkworm.

The DNA sequence encoding a spider silk polypeptide or an analog thereofmay be inserted into the genomic locus for the silkworm silk lightfibroin gene, and/or the silkworm silk heavy fibroin gene. Or, a firstDNA sequence encoding a spider silk fibroin, or analog thereof, may beinserted into the genomic locus for the silkworm silk light fibroingene, and a second DNA encoding a second (i.e., distinct) spider silkfibroin, or analog thereof, may be inserted into the locus for thesilkworm silk heavy fibroin gene the silkworm. Thus, in some cases, aplurality of spider silk polypeptides may be used, such that differentsilkworm transformants express different spider silk polypeptides, oranalogs thereof. Embodiments of the present invention therefore cancombine the best of both producers, the expression machinery of theBombyx silkworm, and the silk produced by the spider.

Other embodiments and further details regarding various aspects of thepresent invention are set forth in the following description and claims.It is to be understood that the invention is not limited in itsapplication to the details set forth in the following description andclaims, but is capable of other embodiments and of being practiced orcarried out in various ways.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a flow-chart of a method to transform silkworms with arecombinant DNA construct that comprises a silkworm silk/spider silkfusion gene by homologous recombination in accordance with oneembodiment of the present invention.

FIG. 2 shows a schematic comparison of the structural organization ofthe Bombyx mori fibroin gene and the synthetic spider silk gene of theinvention.

FIG. 3 shows a schematic representation of the method used to introducethe spider silk gene into Bombyx silkworms.

FIGS. 4A and 4B show a DNA sequence of a spider silk derived internalrepeat segment in accordance with alternate embodiments of the presentinvention; FIG. 4A shows the DNA sequence (SEQ ID NO:1) and amino acidsequence (SEQ ID NO: 2) of a spider silk internal repeat comprising aspider silk analog polypeptide of the present invention; FIG. 4Billustrates the mutation made to an engineered construct of nativespider silk DNA sequences (SEQ ID NO: 3) encoding an alternate spidersilk polypeptide (SEQ ID NO: 4) so as to generate nucleotides 1 to 63 ofSEQ ID NO: 1 encoding amino acids 1 to 21 of SEQ ID NO: 2; FIG. 4C showsan illustration of how a plurality of internal repeat segments, each ofwhich comprise a plurality of beta sheet (dark regions) and alpha helixdomains (light regions), may be combined to generate a spider silkanalog polypeptide.

FIG. 5 shows the DNA sequence (SEQ ID NO: 5) of a 5′ end of a Bombyxmori silk gene that encodes the first exon (SEQ ID NO: 6) and a portionof the second exon (SEQ ID NO: 7) and that may be used to create aspider silk/silkworm silk fusion construct for insertion at a silkwormheavy chain fibroin locus in accordance with one embodiment of thepresent invention. The start and end of the intron is shown as boldfont.

FIG. 6 shows the DNA sequence (SEQ ID NO: 8) of a 3′ end of a Bombyxmori silk gene that encodes the C-terminal portion of the heavy chain(SEQ ID NO: 9) and that may be used to create a spider silk/silkwormsilk fusion construct for insertion at a heavy chain fibroin locus inaccordance with one embodiment of the present invention.

FIG. 7 shows the DNA sequence of an Antheraea pernyi (silkworm) 5′homologous segment (SEQ ID NO: 10) that encodes the first exon (SEQ IDNO: 11) and a portion of the second exon (SEQ ID NO: 12) and that may beused to create a spider silk/silkworm silk fusion construct forinsertion at a heavy chain fibroin locus in accordance with oneembodiment of the present invention. The start and end of the intron isshown as bold font.

FIG. 8 shows the DNA sequence of an Antheraea pernyi (silkworm) 3′homologous segment (SEQ ID NO: 13) that encodes the C-terminal portionof the heavy chain (SEQ ID NO: 14) and that may be used to create aspider silk/silkworm silk fusion construct for insertion at a heavychain fibroin locus in accordance with one embodiment of the presentinvention.

FIG. 9 shows the DNA sequence of an Antheraea yamamai (silkworm) 5′homologous segment (SEQ ID NO: 15) that encodes the first exon (SEQ IDNO: 16) and a portion of the second exon (SEQ ID NO: 17) and that may beused to create a spider silk/silkworm silk fusion construct forinsertion at a heavy chain fibroin locus in accordance with oneembodiment of the present invention. The start and end of the intron isshown as bold font.

FIG. 10 shows the DNA sequence of an Antheraea yamamai (silkworm) 3′homologous segment (SEQ ID NO: 18) that encodes the C-terminal portionof the heavy chain (SEQ ID NO: 19) and that may be used to create aspider silk/silkworm silk fusion construct for insertion at a heavychain fibroin locus in accordance with one embodiment of the presentinvention.

FIG. 11 shows the DNA sequence (SEQ ID NO: 20) encoding a polypeptide(SEQ ID NO: 21) for the 5′ end of a Bombyx mori light fibroin silk geneand that may be used to create a spider silk/silkworm silk fusionconstruct for insertion at a light chain fibroin locus in accordancewith one embodiment of the present invention.

FIG. 12 shows a DNA sequence (SEQ ID NO: 22) encoding a polypeptide (SEQID NO: 23) for the 3′ end of a Bombyx mori light fibroin silk gene andthat may be used to create a spider silk/silkworm silk fusion constructfor insertion at a light chain fibroin locus in accordance with oneembodiment of the present invention. The polyA recognition site is shownas bold font.

FIG. 13 shows a gene assembly route to generate recombinant plasmidconstructs that include multiple numbers of the spider silk analogsequence, i.e., the spider silk internal repeat (I) linked to thesilkworm 5′ sequence (5′), and a silkworm 3′ sequence (3′) in accordancewith one embodiment of the present invention.

FIG. 14 shows a gene assembly route to generate recombinant plasmidconstructs that include multiple numbers (z) of a spider silk analogsequence, i.e., a spider silk internal repeat (I), linked to thesilkworm 5′ sequence (5′), and a silkworm 3′ sequence (3′), andincluding an eGFP marker gene (E) in accordance with one embodiment ofthe present invention.

FIG. 15 shows a DNA sequence (SEQ ID NO: 24) that encodes a Bombyxfibroin intron GFP insert polypeptide (SEQ ID NO: 25) in accordance withone embodiment of the present invention.

FIG. 16 shows a DNA sequence (SEQ ID NO: 26) encoding 5′ Bombyx morihomologous sequence with a knockout insertion and associatedpolypeptides SEQ ID NOS: 27, 28 and 29 in accordance with one embodimentof the present invention.

FIG. 17 shows the DNA sequences of a recombinant DNA construct (SEQ IDNO: 30) that encodes from 5′ to 3′—a Bombyx mori silkworm 5′ sequence(5′), one repeat of a spider silk analog (I), and a silkworm 3′ sequence(3′) in accordance with one embodiment of the present invention.

FIG. 18 shows the DNA sequences of a recombinant DNA construct (SEQ IDNO: 31) that encodes from 5′ to 3′—a Bombyx mori silkworm 5′ sequence(5′); one repeat of a spider silk analog (I); a green fluorescentprotein (GFP) polypeptide (E) and a silkworm 3′ sequence (3′) inaccordance with one embodiment of the present invention.

FIG. 19 shows the DNA sequences of a recombinant DNA construct (SEQ IDNO: 32) that encodes from 5′ to 3′—a Bombyx mori silkworm 5′ sequence(5′), two repeats of a spider silk analog (I₂), and a silkworm 3′sequence (3′) in accordance with one embodiment of the presentinvention.

FIG. 20 shows the DNA sequences of a recombinant DNA construct (SEQ IDNO: 33) that encodes from 5′ to 3′—a Bombyx mori silkworm 5′ sequence(5′), two repeats of a spider silk analog (I₂), a green fluorescentprotein (GFP) polypeptide (E), and a silkworm 3′ sequence (3′) inaccordance with one embodiment of the present invention.

FIG. 21 shows the DNA sequences of a recombinant DNA construct (SEQ IDNO: 34) that encodes from 5′ to 3′—a Bombyx mori silkworm 5′ sequence(5′), three repeats of a spider silk analog (13), and a silkworm 3′sequence (3′) in accordance with one embodiment of the presentinvention.

FIG. 22 shows the DNA sequences of a recombinant DNA construct (SEQ IDNO: 35) that encodes from 5′ to 3′—a Bombyx mori silkworm 5′ sequence(5′), three repeats of a spider silk analog (13), a green fluorescentprotein (GFP) polypeptide (E), and a silkworm 3′ sequence (3′) inaccordance with one embodiment of the present invention.

FIG. 23 shows a comparison of the strength of wild-type (wild type)silkworm silk and the silk of the progeny of silkworms transformed withan embodiments of a construct of the invention (i.e., SEQ ID NO: 32)(transformed); for the transformed silkworms, a portion of the eggs fromsilkworms that had been injected with the recombinant DNA and then matedwere tested for the presence of the altered silk gene (i.e., the spidersilk construct) and silk was isolated from cocoons that developed from aremainder of the eggs.

FIGS. 24A and 24B shows a schematic representation of the results of aPCR of DNA extracts from second generation egg masses transformed with aplasmid containing a 5′-I₂-3′ insert (i.e., SEQ ID NO: 32). The DNA fromabout 75 eggs from each mating was pooled and PCR conducted using aprimer internal to the 3′ non-repetitive section of the native silkwormheavy fibroin gene, and a primer specific to either the synthetic spidersilk derived internal repetitive region (i.e., spider silk analog) (FIG.24A) or a primer specific to the native silkworm heavy fibroin gene(FIG. 24B). The PCR products from random samples (14-18, 20) werecompared to PCR products generated with DNA corresponding to the plasmidwith the silkworm/spider silk gene of SEQ ID NO: 32 (P) and to Bombyxmori genomic DNA (G).

DETAILED DESCRIPTION OF THE INVENTION Definitions

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g. 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10. Additionally, anyreference referred to as being “incorporated herein” is to be understoodas being incorporated in its entirety.

It is further noted that, as used in this specification, the singularforms “a,” “an,” and “the” include plural referents unless expressly andunequivocally limited to one referent. The term “or” is usedinterchangeably with the term “and/or” unless the context clearlyindicates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Practitioners are particularly directed to Current Protocols inMolecular Biology (Ansubel) for definitions and terms of the art.Abbreviations for amino acid residues are the standard 3-letter and/or1-letter codes used in the art to refer to one of the 20 common L-aminoacids.

The term “recombinant” as used herein in relation to a polynucleotideintends a polynucleotide of semisynthetic, or synthetic origin, orencoded by cDNA or genomic DNA (“gDNA”) such that it is not entirelyassociated with all or a portion of a polynucleotide with which it isassociated in nature.

As used herein, the term “polypeptide” refers to a polymer of aminoacids and does not refer to a specific length of the product. Thus,peptides, oligopeptides, and proteins are included within the definitionof polypeptide. This term also does not exclude post-expressionmodifications of the polypeptide, for example, glycosylations,acetylations, phosphorylations and the like. Included within thedefinition are, for example, polypeptides containing one or more analogsof an amino acid (including, for example, unnatural amino acids),polypeptides with substituted linkages, as well as other modificationsknown in the art, both naturally occurring and non-naturally occurring.As is known in the art, “proteins”, “peptides,” “polypeptides” and“oligopeptides” are chains of amino acids (typically L-amino acids)whose alpha carbons are linked through peptide bonds formed by acondensation reaction between the carboxyl group of the alpha carbon ofone amino acid and the amino group of the alpha carbon of another aminoacid. Typically, the amino acids making up a protein are numbered inorder, starting at the amino terminal residue and increasing in thedirection toward the carboxy terminal residue of the protein.

As used herein, a polypeptide or protein “domain” comprises a regionalong a polypeptide or protein that comprises an independent unit.Domains may be defined in terms of structure, sequence and/or biologicalactivity. In one embodiment, a polypeptide domain may comprise a regionof a protein that folds in a manner that is substantially independentfrom the rest of the protein. Domains may be identified using domaindatabases such as, but not limited to PFAM, PRODOM, PROSITE, BLOCKS,PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.

A “nucleic acid” is a polynucleotide such as deoxyribonucleic acid (DNA)or ribonucleic acid (RNA). The term is used to include single-strandednucleic acids, double-stranded nucleic acids, and RNA and DNA made fromnucleotide or nucleoside analogues. The term “polynucleotide” as usedherein refers to a DNA molecule, a RNA molecule or its complementarystrand thereof. A polynucleotide molecule can be single or doublestranded.

DNA molecules may be identified by their nucleic acid sequences, whichare generally presented in the 5′ to 3′ direction, wherein 5′ and 3′indicate the linkages formed between the 5′-phosphate group of onenucleotide and the 3′-hydroxyl group of the next. For a sequencepresented in the 5′ to 3′ direction, its complement is the DNA strandwhich hybridizes to that sequence according to the Watson-Crick basepairing model. Thus, the sequence of the complement is defined by thesequence of the original strand, such that adenine base-pairs withthymine, and cytosine base-pairs with guanine.

As used herein, a small inhibitory RNA is a double-stranded RNA of about20-30 nucleotides that associates with proteins to form an RNAi-inducedsilencing complex (RISC) that may direct the siRNA to the target RNAsequence. The ds siRNA may then unwind, leaving the antisense strand tosignal degradation of the mRNA sequence by endonucleases andexonucleases. In order to obtain lasting therapeutic effects, the RNAisequence may be expressed long term, preferably under a constitutivepromoter. To obtain dsRNA from a vector, it may be expressed as a shorthairpin RNA (shRNA), in which there is a sense strand, a hairpin loopregion and an antisense strand (Miyagishi et al., J Gene Med 6:715-723,2004).

As used herein, the term “upstream” refers to a residue that isN-terminal to a second residue where the molecule is a protein, or 5′ toa second residue where the molecule is a nucleic acid. Also as usedherein, the term “downstream” refers to a residue that is C-terminal toa second residue where the molecule is a protein, or 3′ to a secondresidue where the molecule is a nucleic acid. Also, the terms “portion”and “fragment” are used interchangeably to refer to parts of apolypeptide, nucleic acid, or other molecular construct.

The term “vector” refers to a nucleic acid molecule that may be used totransport a second nucleic acid molecule into a cell. In one embodiment,the vector allows for replication of DNA sequences inserted into thevector. The vector may comprise a promoter to enhance and/or maintainexpression of the nucleic acid molecule in at least some host cells.Vectors may replicate autonomously (extrachromasomally) or may beintegrated into a host cell chromosome. In one embodiment, the vectormay comprise an expression vector capable of producing a protein or anucleic acid derived from at least part of a nucleic acid sequenceinserted into the vector.

As is known in the art, conditions for hybridizing nucleic acidsequences to each other can be described as ranging from low to highstringency. Generally, highly stringent hybridization conditions referto washing hybrids in low salt buffer at high temperatures.Hybridization may be to filter bound DNA using hybridization solutionsstandard in the art such as 0.5M NaHPO₄, 7% sodium dodecyl sulfate(SDS), at 65° C., and washing in 0.25 M NaHPO₄, 3.5% SDS followed bywashing 0.1×SSC/0.1% SDS at a temperature ranging from room temperatureto 68° C. depending on the length of the probe (see e.g. Ausubel, F. M.et al., Short Protocols in Molecular Biology, 4^(th) Ed., Chapter 2,John Wiley & Sons, N.Y.). For example, a high stringency wash compriseswashing in 6×SSC/0.05% sodium pyrophosphate at 37° C. for a 14 baseoligonucleotide probe, or at 48° C. for a 17 base oligonucleotide probe,or at 55° C. for a 20 base oligonucleotide probe, or at 60° C. for a 25base oligonucleotide probe, or at 65° C. for a nucleotide probe about250 nucleotides in length. Nucleic acid probes may be labeled withradionucleotides by end-labeling with, for example, [gamma-³²P]ATP, orincorporation of radiolabeled nucleotides such as [alpha-³²P]dCTP byrandom primer labeling. Alternatively, probes may be labeled byincorporation of biotinylated or fluorescein labeled nucleotides, andthe probe detected using Streptavidin or anti-fluorescein antibodies.

The terms “identity” or “percent identical” refer to sequence identitybetween two amino acid sequences or between two nucleic acid sequences.Percent identity can be determined by aligning two sequences and refersto the number of identical residues (i.e., amino acid or nucleotide) atpositions shared by the compared sequences. Sequence alignment andcomparison may be conducted using the algorithms standard in the art(e.g. Smith and Waterman, 1981, Adv. Appl. Math. 2:482; Needleman andWunsch, 1970, J. Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc.Natl. Acad. Sci., USA, 85:2444) or by computerized versions of thesealgorithms (Wisconsin Genetics Software Package Release 7.0, GeneticsComputer Group, 575 Science Drive, Madison, Wis.) publicly available asBLAST and FASTA. Also, ENTREZ, available through the National Institutesof Health, Bethesda Md., may be used for sequence comparison. In oneembodiment, the percent identity of two sequences may be determinedusing GCG with a gap weight of 1, such that each amino acid gap isweighted as if it were a single amino acid mismatch between the twosequences. For example, the term at least 90% identical thereto includessequences that range from 90 to 100% identity to the indicated sequencesand includes all ranges in between. Thus, the term at least 90%identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93,93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percentidentical to the indicated sequence. Similarly the term “at least 70%identical includes sequences that range from 70 to 100% identical, withall ranges in between. The determination of percent identity isdetermined using the algorithms described here.

As used herein, “homology” refers to the degree of similarity betweentwo proteins and or nucleic acid sequences. Homologous proteins arethose that are similar in sequence and function. Typically, the sequenceidentity between two homologous sequences will be at least 50%. Also,homologous proteins will have conservative substitutions fornon-identical sequences. In alternate embodiments, the sequence identitybetween two homologous sequences will be at least 60%; or at least 75%;or at least 80%; or at least 90%, or at least 95%, or at least 96%, orat least 97%, or at least 98%, or at least 99%. Also, as used herein,the term “homologue” means a polypeptide having a degree of homologywith the wild-type amino acid sequence. Homology comparisons can beconducted by eye, or more usually, with the aid of readily availablesequence comparison programs. These commercially available computerprograms can calculate percent homology between two or more sequences(e.g. Wilbur, W. J. and Lipman, D. J., 1983, Proc. Natl. Acad. Sci. USA,80:726-730).

As used herein, the term “silkworm” refers to a larvae of any member ofthe Saturniids, more typically the genus Bombyx, especially if usedwithout modifier (e.g. Antheraea silkworm). Less formally, silkworm mayalso refer to adults of Lepidoptera most typically the Saturniids, andespecially the genera Bombyx and Antheraea.

As used herein, the term “spider” refers to air-breathing cheliceratearthropods that have two body segments, eight legs, no chewing parts andthat make silk. Spiders of the present invention are from the Arachnidaclass, the Araneae order, and include for example Nephilidae (especiallyNephila species, like clavipes), and Araneidae such as Araneus andArgiopes, among many other suitable species.

As used herein, “silk” includes proteins and peptides produced byarthropods, typically by spiders, or by Lepidoptera, that displayproperties typical of native silk peptides. Lepidopteran silk generallyis made up of a heavy fibroin polypeptide and a light chain fibroinpeptide that are joined by a disulfide bond. In spiders there are two ormore peptides not joined by a disulfide bond. Thus, silk includesproteinaceous filaments produced by insects or spiders, typically (butnot necessarily) of two or more polypeptides. These may be chemicallylinked, and are typically very long polypeptides.

A native silkworm silk polypeptide is one several proteins orpolypeptides, or fragments thereof, produced by silkworm silk glands. Asused herein, a native silkworm silk polypeptide is a polypeptide havingat least 99% identity to a native silkworm silk heavy and/or lightfibroin polypeptide. For example, silkworm silk comprises and mayconsist of the silk polypeptides produced by members of the genusBombyx.

A native spider silk polypeptide is one of the proteins or polypeptides,or fragments thereof, produced by spider silk glands. As used herein, anative spider silk polypeptide is a polypeptide having at least 99%identity, or in some cases 100% identity, to a native spider silk heavyand/or light fibroin polypeptide. Spider silk is a protein based fiber.It is known for its high strength and elasticity. Each species of spiderproduces several kinds of silk, and the silks vary in sequence betweenthe species. Each of these types of silk is encompassed by the presentinvention. Some of the varieties of silk produced by spiders for whicheither the natural peptides, or peptide analogs are encompassed by themethods, compositions and systems of the invention are: (a) majorampullate silk—a tough, strong, and elastic silk that is used for thewebs and spokes of webs as well as draglines; (b) flagelliform silk—avery stretchy, sticky silk that is used to capture insects; (c)tubiliform silk—a very stiff spider silk that is used to produce eggcases; (d) aciniform silk—a tough and elastic silk that is used to wrapcaptured prey; and (e) minor-ampullate silk—a silk that is somewhat lesstough and elastic compared to dragline silk.

As used herein, an “analog of a spider silk” or an “analog of a spidersilk polypeptide” comprises or consists of a polypeptide having aminoacid domains, such as beta-sheets and alpha helices that are derivedfrom, or homologous to, those domains as found in spider silk proteins.In certain embodiments, a spider silk analog polypeptide is comprised ofpeptide domains that are at least 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, or 98% identical to native spider silk. For example, the spidersilk analog may comprise, or consist of, a sequence made up of aplurality of alternating spider silk beta-sheet sequences and alphahelices as described herein. In certain embodiments, the spider silkpolypeptide may comprise from 4 to 1000, or 4 to 800, or 4 to 500, or 5to 200, or 5 to 100, or 5 to 50, or 6 to 40, or 6 to 30, or 6 to 15 or 6to 12, or about 9 beta-sheet domains. The beta sheet regions maycomprise a plurality of consecutive alanine residues, or a plurality ofother amino acids that can form hydrogen bonds and that are typicallyarranged in consecutive order in beta sheet regions, and may range fromabout or 3 to 50, or 4 to 40, or 4 to 30, or 4 to 15, or 4 to 12, or 6to 10, or about 9 consecutive hydrogen bonding amino acids (e.g.,(Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala). In certain embodiments, thespider silk polypeptide may comprise from 4 to 1000, or 4 to 800, or 4to 500, or 5 to 200, or 5 to 100, or 5 to 50, or 6 to 40, or 6 to 30, or6 to 15 or 6 to 12, or about 9 or 10 alpha helix domains. The alphahelix domains may comprise a plurality of glycine residues interspersedwith other amino acids (e.g., Q, Y, L, S. R, A or P) typically found inalpha helix domains, and may range from about 4 to 200, or 5 to 100, 5to 50, or 6 to 45, or 12 to 40, or 12 to 45 amino acids in length.

In certain embodiments, the spider silk peptide domains are derivedfrom, i.e., are at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 98%identical to spider silk fibroin sequences. Also, a spider silk analogmay comprise a single polypeptide having a mixture of different spidersilk polypeptide domains, or analogs thereof, either from the same ordifferent species. Example domains that may be used to generate a spidersilk polypeptide of SEQ ID NO: 2 include the following peptides:LGGQGAAAAAAAAAGGGGQGG (SEQ ID NO 36), GYGGLGSQAGRGG (SEQ ID NO: 37),LGGQGGGQ (SEQ ID NO: 38), GSGRGG (SEQ ID NO: 39), LGGQGAAAAAAAAAGAGGQGG(SEQ ID NO 40), and LGGQGAGQ (SEQ ID NO: 41).

The analogs may further include peptides having one or more peptidemimetics, also known as peptoids, that possess the bioactivity of theprotein. Included within the definition are also polypeptides containingone or more amino acid analogs (including, for example, unnatural aminoacids, etc.), polypeptides with substituted linkages, as well as othermodifications known in the art, both naturally occurring andnon-naturally occurring. The term polypeptide also does not excludepost-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations and the like.

Spider silk analogs may be generated using molecular techniques. Forexample, PCR mutagenesis of DNA encoding the spider silk peptide analogscan be used. Or RNA based mutagenesis techniques may be used. An exampleof a PCR technique for making mutations in DNA is described in WO92/22653. Another method for making analogs, muteins, and derivatives,is cassette mutagenesis based on the technique described by Wells, Gene,(1985) 34:315. Or, chemical modification of the peptides may beperformed.

Thus, the analogs of spider silk polypeptides may contain amino acidsubstitutions, deletions, or insertions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acid residues such as to alter aglycosylation site, a phosphorylation site, an acetylation site, or tominimize misfolding by substitution or deletion of one or more cysteineresidues that are not necessary for function.

As used herein, the term “conserved residues” refers to amino acids thatare the same among a plurality of proteins having the same structureand/or function. A region of conserved residues may be important forprotein structure or function. Thus, contiguous conserved residues asidentified in a three-dimensional protein may be important for proteinstructure or function. To find conserved residues, or conserved regionsof 3-D structure, a comparison of sequences for the same or similarproteins from different species, or of individuals of the same species,may be made. Conservative amino acid substitutions are generally thosethat preserve the general charge, hydrophobicity/hydrophilicity and/orsteric bulk of the amino acid substituted, for example, substitutionsbetween the members of the following groups are conservativesubstitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln,Ser/Cys/Thr and Phe/Trp/Tyr.

As used herein, a homozygous transformant includes silkworms that haveboth native silkworm light fibroin or heavy fibroin loci replaced by aDNA that encodes for a spider silk gene or analog thereof, and includetransformants in which the spider silk sequences at each of the loci arenot the same, but encode distinct spider silk polypeptides or analogsthereof.

An “expression vector” is a polynucleotide that is operable in a desiredhost cell and capable of causing the expression of a gene of interest inthat host cell.

A “regulatory sequence” refers to a polynucleotide sequence that isnecessary for regulation of expression of a coding sequence to which thepolynucleotide sequence is operably linked. The nature of suchregulatory sequences may differ depending upon the host organism. Suchregulatory sequences generally include, for example, a promoter, and/ora transcription termination sequence. The term “regulatory sequence” mayalso include additional components the presence of which areadvantageous, for example, a secretory leader sequence for secretion ofthe polypeptide attached thereto.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence is “operably linked” to a codingsequence when it is joined in such a way that expression of the codingsequence is achieved under conditions compatible with the regulatorysequence. Operably linked sequences may have additional nucleotides (oramino acids in a peptide) positioned between the two components ofinterest.

As used herein, “terminators” are regulatory sequences, such aspolyadenylation and transcription termination sequences, located 3′ ordownstream of the stop codon of the coding sequences.

As used herein, “recombinant host cells,” “host cells,” “cells,” “cellcultures,” and other such terms denote, for example silkworm eggs orcells derived therefrom that have been used as recipients forintroduction of recombinant vector or other transfer DNA, and includethe progeny of the cell that has been transformed.

“Transformation” or “transfection,” as used herein, refers to thetransfer of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the transfer, which can be, for example, byinfection, direct uptake, transduction, F-mating, injection,microinjection or electroporation. The exogenous polynucleotide may bemaintained as a non-integrated vector, for example, in some cases, aplasmid, or alternatively, may be integrated into the host genome.

“Purified” and “isolated” in reference to a polypeptide or a nucleotidesequence means that the indicated molecule is present in substantialabsence of other biological macromolecules of the same species or type.In alternate embodiments, the term “purified” as used herein refers toat least 75% by weight; or at least 85% by weight, or at least 95% byweight or at least 98% by weight, of biological macromolecules of thesame type.

An adjuvant is a chemical or biological agent that modifies or enhancesthe effect of other agents (e.g., drugs, vaccines, plasmids) whilehaving few if any direct effects when given by themselves. Roughlyanalogous to chemical catalysts, they may have the effect of enhancingthe entry into a host cell of DNA segments or plasmids, or enhancing therecombination of such DNA with the host cell DNA.

Methods, Compositions and Systems for Production of Recombinant Silk

Embodiments of the present invention comprise methods, compositions andsystems for producing high-quality silk. The present invention may beembodied in a variety of ways.

In certain embodiments, the present invention comprises methods,compositions and systems for expressing a recombinant silk polypeptideby inserting DNA encoding the recombinant silk into the genomic locusfor a native silk polypeptide in a silk-producing organism. In certainembodiments, the present invention comprises methods, compositions,and/or systems to express in a first organism, a silk polypeptide, oranalog thereof, from a second organism. In certain embodiments, arecombinant DNA construct comprising sequences that encode for a silkpolypeptide, or analog thereof, from the second organism is insertedinto the genome of the first organism such that the sequences encodingfor the silk polypeptide, or an analog thereof, from the second organismare expressed in the first organism. In certain embodiments, the DNAconstruct comprising sequences that encode for a silk polypeptide, or ananalog thereof, from the second organism is inserted into the genome ofthe first organism such that the sequences encoding for the silkpolypeptide, or analog thereof, from the second organism replace genomicsequences for native silk in the first organism, such that the silkpolypeptide from the second organism replaces a silk polypeptide made bythe first organism.

In one embodiment, the first organism is a silkworm. For example, Bombyxmori silkworms may be used as the first organism. Or, other types ofsilk-producing organisms may be used, such as Antheraea pernyi,assamensis, or yamami; Samia cynthia; or Gonometa species, such aspostica or rufobrunnea; among other suitable species.

In certain embodiments, the silk polypeptide, or analog thereof, isderived from spider silk. Or silk from other organisms such asTrichoptera may be used. Thus, certain embodiments of the presentinvention comprise replacing silkworm silk genomic sequences withsequences that encode for a spider silk polypeptide or analog thereof.For example, in certain embodiments, the silk polypeptide is spiderdragline silk or an analog thereof. Or, other types of spider silk, suchas: (a) major ampullate silk; (b) flagelliform silk; (c) tubiliformsilk; (d) aciniform silk; or (e) minor-ampullate silk, or analogsthereof, may be produced. It is understood, however, that in certainembodiments, organisms other than silkworm may be used as silkbioreactors, and peptides derived from organisms other than spiders maybe used in the methods, systems and compositions of the presentinvention.

The spider silk polypeptide used in the methods, compositions andsystems of the present invention may be a native spider silkpolypeptide, or may be an analog of a native spider silk polypeptide. Incertain embodiments, an analog of a spider silk polypeptide comprises apeptide having amino acid domains, such as beta sheets and alpha helicesthat are derived from, or homologous to, those domains as found inspider silk proteins. In certain embodiments, the spider silkpolypeptide domains are derived from spider silk fibroin sequences. Or amixture of spider silk polypeptide domains may be used.

For example, in certain embodiments, the present invention providesmethods, compositions and systems for expression of native spider silk(e.g., such as dragline silk), or analogs of spider silk in silkworms(e.g., Bombyx mori). In certain embodiments, the present inventionreduces or eliminates the dilution of spider silk by natural Bombyx silkin the transformed Bombyx mori.

Thus, embodiments of the present invention may comprise methods to makea silkworm that is capable of producing a silk comprising a spider silkpolypeptide, or an analog of a spider silk polypeptide. In certainembodiments, the spider silk polypeptide is encoded by a recombinantDNA.

For example, in one embodiment, the invention may comprise a methodcomprising inserting a DNA sequence coding for at least a portion of aspider silk fibroin polypeptide, or an analog of a spider silk fibroinpolypeptide, into the genome of a silkworm. In certain embodiments, thetransformed silkworm expresses a polypeptide comprising a spider silkfibroin, or an analog thereof, and does not express substantial amountsof the light chain and/or heavy chain native silkworm silk fibroinpolypeptide.

In certain embodiments, the native silkworm fibroin genes aregenetically modified such that at least one native silkworm fibroinpeptide that is normally used to form silkworm silk is not expressed toa level that is sufficient to generate silkworm silk. The geneticmodification of the silkworm fibroin gene or genes may comprise the stepof eliminating expression of at least one native silkworm fibroin gene.Genetic techniques that are known in the art for knocking out geneexpression may be used to eliminate expression of the native (i.e.,wildtype) silkworm gene or genes.

For example, homologous recombination may be used to knock-out thenative silkworm gene and/or to replace the silkworm gene with arecombinant DNA encoding a spider silk polypeptide or analog thereof.Embodiments of methods and constructs that may be used to generatesilkworm knockouts are provided in the Examples herein. In this way, thespider silk polypeptide or analog thereof, will be expressed, but thenative silkworm silk polypeptide is not expressed in substantialamounts. In certain embodiments, a plasmid vector is used to insert arecombinant DNA construct encoding a spider silk polypeptide, or analogthereof, into the silkworm genome. For example, a baculovirus vector, orother vectors known in the art, may be used to insert the spider silksequences into the silkworm genome.

In some embodiments, the DNA encoding for a spider silk peptide, oranalog thereof, may be inserted anywhere in the silkworm genome suchthat expression of the spider silk polypeptide occurs. In thisembodiment, however, there may be production of both the native silkwormpolypeptide and the spider silk polypeptide or analog thereof. Thus, toprevent the native silkworm silk polypeptide (e.g., heavy or lightsilkworm fibroin) from competing with the recombinant spider silkpolypeptide, this embodiment may include the step of knocking outexpression of the native silkworm gene as discussed herein. In that way,the spider silk polypeptide, or analog thereof is preferentiallyexpressed.

In other embodiments, site-specific recombination is used such that theDNA encoding a spider silk polypeptide or analog thereof is insertedinto the silkworm silk genomic locus so as to replace the gene encodingeither the light or heavy chain silkworm fibroin (e.g., a spider silkfibroin replacing a silkworm heavy fibroin). In some embodiments, thegene introduced into the silkworm is a fusion gene that encodes apolypeptide comprising spider silk and silkworm silk sequences.

Thus, in certain embodiments, the methods of the present invention maybe used to introduce spider silk sequences, or analogs thereof, intosilkworms, or other natural producers of silk. In one embodiment, themethods and/or compositions are used to replace a native silk gene(e.g., heavy or light fibroin) with a recombinant gene that encodes forthe desired silk analog polypeptide.

For example, in one embodiment, the method may comprise a method to makea silkworm that is capable of producing a silk comprising a spider silkpolypeptide, or an analog of a spider silk polypeptide, where the methodcomprises ligating a DNA sequence coding for at least a portion of aspider silk fibroin polypeptide, or an analog of a spider silk fibroinpolypeptide, between at least a portion of the 5′ and 3′ ends of asilkworm fibroin gene to generate a fusion gene construct (i.e., a fusedgene) having a sequence that encodes for a polypeptide comprising bothspider silk fibroin and silkworm silk fibroin amino acid sequences(i.e., a spider silk/silkworm silk gene). In certain embodiments, the 5′and 3′ ends of a silkworm fibroin gene are long enough such that whenthe recombinant DNA is inserted into the nucleus of a silkworm,site-specific homologous recombination can occur such that the fusiongene replaces the native silk worm gene.

Thus, in certain embodiments, the present invention comprises a methodto make a silkworm that is capable of producing a silk comprising aspider silk polypeptide, or analog thereof, comprising the steps ofligating a DNA sequence coding for at least a portion of a spider silkfibroin polypeptide or an analog thereof, between at least a portion ofthe 5′ and 3′ ends of a silkworm fibroin gene to generate a fusion geneconstruct having a sequence that encodes for a polypeptide comprisingboth spider silk fibroin and silkworm silk fibroin amino acid sequences,wherein the 5′ and 3′ ends of a silkworm fibroin gene are long enough toallow for site-specific homologous recombination such that when thefusion gene is inserted into a silkworm, it is able to replace thenative gene present in the silkworm.

The method may further comprise transforming a first population ofsilkworms with a fusion gene construct encoding a spider silk fibroingene, or analog thereof, and transforming a second population ofsilkworms with a fusion gene construct encoding a second (i.e.,different) spider silk fibroin gene. The DNA sequence encoding a spidersilk polypeptide or an analog thereof may be inserted into the genomiclocus for the silkworm silk light fibroin gene, and/or the silkworm silkheavy fibroin gene. Or, a first DNA sequence encoding a spider silkfibroin, or analog thereof, may be inserted into the genomic locus forthe silkworm silk light fibroin gene, and a second DNA encoding a second(i.e., distinct) spider silk fibroin, or analog thereof, may be insertedinto the locus for the silkworm silk heavy fibroin gene. Thus, in somecases, a plurality of spider silk polypeptides may be used, such thatdifferent silkworm transformants express different spider silkpolypeptides, or analogs thereof.

The method may comprise breeding the recombinant silkworms to generatesilkworms that are homozygous at a fibroin loci for the fused geneconstruct. In alternate embodiments, the methods may thus comprisebreeding the recombinant silkworms to generate silkworms that are eitherhomozygous at a single silkworm loci and/or homozygous at two or moresilkworm loci and/or that contain a spider silk/silkworm silk fusionconstruct at multiple loci as described herein. In certain embodimentsat least one of light fibroin loci and one heavy fibroin chain in arecipient silkworm are replaced by a fusion gene construct encoding aspider silk fibroin polypeptide or an analog of a spider silk fibroinpolypeptide. For example in certain embodiments, a silkworm having afirst DNA sequence encoding a spider silk fibroin, or analog thereof,inserted into the genomic locus for the silkworm silk light fibroingene, can be bred with a second silkworm having the same or a second DNAencoding a second (i.e., distinct) spider silk fibroin, or analogthereof, inserted into the locus for the silkworm silk heavy fibroingene the silkworm so as to generate silkworms that express spider silkpolypeptides, or analogs thereof, at both the light and heavy chain lociin a recipient silkworm.

In other embodiments, the present invention comprises compositionscomprising a DNA sequence encoding a recombinant spider silk fibroingene or a portion of a recombinant spider silk fibroin gene that encodesfor an analog of a spider silk polypeptide.

For example, in one embodiment, the present invention comprises anisolated and/or recombinant DNA molecule having a nucleic acid sequenceencoding an analog of a spider silk polypeptide. In one embodiment, therecombinant construct encodes both spider silk fibroin and silkworm silkfibroin amino acid sequences. In this embodiment, the recombinantconstruct may comprise a fusion gene. For example in one embodiment, thepresent invention comprises a composition comprising a DNA sequenceencoding both spider silk fibroin and silkworm silk fibroin amino acidsequences, the composition comprising a recombinant DNA construct codingfor at least a portion of a spider silk fibroin polypeptide, or ananalog of a spider silk fibroin polypeptide, or a biological equivalentthereof, positioned between at least a portion of the 5′ and 3′ ends ofa silkworm fibroin gene to generate a fusion gene construct having asequence that encodes for a polypeptide comprising both spider silkfibroin and silkworm silk fibroin amino acid sequences.

The constructs of the present invention may allow for a native silkwormgene for either light or heavy chain fibroin to be replaced by aconstruct encoding the spider silk fibroin polypeptide or analogthereof. For example, the present invention may comprise a DNA moleculeencoding for both spider silk fibroin and silkworm silk fibroinsequences. In certain embodiments, the recombinant DNA construct encodesfor at least a portion of a spider silk fibroin polypeptide or an analogthereof, positioned between at least a portion of the 5′ and 3′ ends ofa silkworm fibroin gene to generate a fusion gene construct having asequence that encodes for a polypeptide comprising both spider silkfibroin and silkworm silk fibroin, wherein the 5′ and 3′ ends of asilkworm fibroin gene are long enough to allow for site-specifichomologous recombination to occur, such that when the DNA construct isinserted into the nucleus of a silkworm, it is able to replace thenative gene present in the silkworm.

In other embodiments, the present invention comprises systems, such as asilkworm or a silkworm egg or larvae, that expresses or encodes a spidersilk polypeptide sequences or an analog thereof.

In one embodiment, the silkworms or silkworm eggs or larvae, maycomprise a recombinant DNA construct comprising at least a portion of aspider silk fibroin polypeptide, or an analog of a spider silk fibroinpolypeptide, or a biological equivalent thereof, operably linked (e.g.,positioned between) at least a portion of the 5′ and 3′ ends of asilkworm fibroin gene to generate a fusion gene construct having asequence that encodes for a polypeptide comprising both spider silkfibroin and silkworm silk fibroin amino acid sequences. In certainembodiments, the silkworm 5′ and 3′ sequences are directly linked to thesequences encoding a spider silk polypeptide or analog thereof. Incertain embodiments, the transformed silkworm is generated using arecombinant DNA construct encoding for at least a portion of a spidersilk fibroin polypeptide or an analog thereof, positioned between atleast a portion of the 5′ and 3′ ends of a silkworm fibroin gene togenerate a fused gene having a sequence that encodes for a polypeptidecomprising both spider silk fibroin and silkworm silk fibroin, whereinthe 5′ and 3′ ends of a silkworm fibroin gene are long enough to allowfor site-specific homologous recombination to occur when the DNAconstruct is inserted into the nucleus of a silkworm.

The spider silk polypeptide, or analog thereof, used in the methods,compositions and systems of the present invention may comprise orconsist of a polypeptide having amino acid domains, such as beta-sheetsand alpha helices that are derived from, or homologous to, those domainsas found in spider silk proteins. In certain embodiments, a spider silkanalog polypeptide is comprised of peptide domains that are at least50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, or 98% identical to nativespider silk.

For example, the spider silk polypeptide, or analog thereof, maycomprise, or consist of, a plurality of alternating beta-sheet sequencesand alpha helices that are derived from spider silk sequences asdescribed herein. In some embodiments, the spider silk analogpolypeptide comprises a plurality of such domains, wherein each domaincomprises a plurality of alternating beta sheet domains and alpha helixdomains. In an embodiment, the domains comprising a plurality ofalternating beta sheet domains and alpha helix domains comprises a unitthat may be designated as a spider silk analog internal repeat (I).

For example, in certain embodiments, the spider silk polypeptide oranalog thereof may comprise a single spider silk internal repeat domain(I). Or, the spider silk polypeptide or analog thereof may comprise aplurality of the same or different single spider silk internal repeatdomains (e.g., IA, IB, IC, ID and the like) linked end to end in variousarrangements as described in more detail herein. For example, thevarious internal repeat regions may vary from each other in havingdifferent beta sheet regions (i.e., domains) and/or different alphahelix domains and/or different numbers of each of these domains.

In certain embodiments, the spider silk polypeptide, or a spider silkpolypeptide internal repeat (I), may comprise from 4 to 1000, or 4 to800, or 4 to 500, or 5 to 200, or 5 to 100, or 5 to 50, or 6 to 40, or 6to 30, or 6 to 15 or 6 to 12, or about 9 beta-sheet domains. The betasheet regions may comprise a plurality of consecutive alanine residues,or a plurality of other amino acids that can form hydrogen bonds andthat are typically arranged in consecutive order in beta sheet regions,and may range from about or 3 to 50, or 4 to 40, or 4 to 30, or 4 to 15,or 4 to 12, or 6 to 10, or about 9 consecutive hydrogen bonding aminoacids (e.g., (Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala-Ala). In certainembodiments, the spider silk polypeptide may comprise from 4 to 1000, or4 to 800, or 4 to 500, or 5 to 200, or 5 to 100, or 5 to 50, or 6 to 40,or 6 to 30, or 6 to 15 or 6 to 12, or about 9 or 10 alpha helix domains.The alpha helix domains may comprise a plurality of glycine residuesinterspersed with other amino acids (e.g., Q, Y, L, S. R, A or P)typically found in alpha helix domains, and may range from about 4 to200, or 5 to 100, 5 to 50, or 6 to 45, or 12 to 40, or 12 to 45 aminoacids in length. Thus, in alternate embodiments, the spider silk analogmay comprise a sequence made up of about 4 to 1000, or 4 to 800, or 4 to500, or 5 to 200, or 5 to 100, or 5 to 50, or 6 to 40, or 6 to 30, or 6to 15 or 6 to 12, or about 9 spider silk beta-sheet domains alternatingwith about 4 to 1000, or 4 to 800, or 4 to 500, or 5 to 200, or 5 to100, or 5 to 50, or 6 to 40, or 6 to 30, or 6 to 15 or 6 to 12, or about9 to10 spider silk alpha helix domains.

For example, in certain embodiments of the methods, compositions andsystems of the present invention, the spider silk peptide domains arederived from, i.e., are at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%,or 98% identical to spider silk fibroin sequences. Also, a spider silkanalog may comprise a single polypeptide having a mixture of differentspider silk polypeptide domains, or analogs thereof, either from thesame or different species. Thus, in certain embodiments, the fusion geneconstruct comprises a nucleic acid that encodes for a peptide having theamino acid sequence as set forth in at least one of SEQ ID NO: 36, SEQID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO:41. Or, the fusion gene construct may comprise a nucleic acid thatencodes for a spider silk analog peptide having the amino acid sequenceas set forth in SEQ ID NO: 2.

Example domains that may be used to generate a spider silk polypeptideanalog include the following peptides: LGGQGAAAAAAAAAGGGGQGG (SEQ ID NO36), GYGGLGSQAGRGG (SEQ ID NO: 37), LGGQGGGQ (SEQ ID NO: 38), GSGRGG(SEQ ID NO: 39), LGGQGAAAAAAAAAGAGGQGG (SEQ ID NO 40), and LGGQGAGQ (SEQID NO: 41). Thus, as discussed in more detail herein, the spider silksequences, or an analog thereof, (e.g., SEQ ID NO: 2) of the methods,compositions and systems of the present invention may comprise a singleinternal repeat unit (I) which is made up of smaller units: SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40 and/orSEQ ID NO: 41. However, various embodiments of the methods, compositionsand systems of the present invention comprise one or more of theseinternal repeats ligated to various silkworm 5′ and 3′ ends. Also, therecombinant constructs may comprise a reporter gene, such as a greenfluorescent protein to facilitate determination of whether successfultransformation has occurred.

Thus, in certain embodiments the construct may comprise at least one ofthe following:

-   -   (a) a recombinant DNA construct (SEQ ID NO: 30) that encodes        from 5′ to 3′—a Bombyx mori silkworm 5′ sequence (5′), one        repeat of a spider silk analog (I), and a silkworm 3′ sequence        (3′);    -   (b) a recombinant DNA construct (SEQ ID NO: 31) that encodes        from 5′ to 3′—a Bombyx mori silkworm 5′ sequence (5′); one        repeat of a spider silk analog (I); a green fluorescent protein        (GFP) polypeptide (E) and a silkworm 3′ sequence (3′);    -   (c) a recombinant DNA construct (SEQ ID NO: 32) that encodes        from 5′ to 3′—a Bombyx mori silkworm 5′ sequence (5′), two        repeats of a spider silk analog (I₂), and a silkworm 3′ sequence        (3′);    -   (d) a recombinant DNA construct (SEQ ID NO: 33) that encodes        from 5′ to 3′—a Bombyx mori silkworm 5′ sequence (5′), two        repeats of a spider silk analog (I₂), a green fluorescent        protein (GFP) polypeptide (E), and a silkworm 3′ sequence (3′);    -   (e) a recombinant DNA construct (SEQ ID NO: 34) that encodes        from 5′ to 3′—a Bombyx mori silkworm 5′ sequence (5′), three        repeats of a spider silk analog (I), and a silkworm 3′ sequence        (3′); or    -   (f) a recombinant DNA construct (SEQ ID NO: 35) that encodes        from 5′ to 3′—a Bombyx mori silkworm 5′ sequence (5′), three        repeats of a spider silk analog (13), a green fluorescent        protein (GFP) polypeptide (E), and a silkworm 3′ sequence (3′).

Additional sequences having multiple copies of the internal repeat (I)ranging from 2 to 1000 or more may be generated using the sequencedisclosed herein as embodiments of the spider silk analog constructs ofthe present invention. Or sequences at least 70%, or 75%, or 80%, or85%, or 90%, or 95%, or 96%, or 97%, or 98% or 99% identical to each ofthese sequences may be used.

As detailed herein, the DNA encoding a spider silk polypeptide, oranalog thereof, used in the methods, compositions and systems of thepresent invention may be operably linked to silkworm sequences so as topromote site-specific insertion of the DNA encoding the spider silkpeptide or analog thereof into the silkworm genome. The length of the 5′and 3′ ends of the silkworm gene should be long enough such thathomologous recombination between the recombinant spider silk/silkwormsilk fusion gene can occur. In alternate embodiments, the 5′ and/or 3′ends may comprise at least 200, or at least 300, or at least 400, or atleast 500, or at least 600, or at least 700, or at least 800, or atleast 900, or at least 1000, or at least 1100, or at least 1200, or atleast 1300, or at least 1400, or at least 1500, or at least 1600, or atleast 1700, or at least 1800, or at least 1900, or at least 2000nucleotides of the silkworm genomic DNA. Also, in alternate embodiments,the 5′ and/or 3′ ends may comprise about 100 to 3000, or 200 to 2500, or300 to 2000, or 400 to 1800, or 500 to 1600, or 500 to 1500, or 500 to1400, or 500 to 1200, or 500 to 1100 nucleotides of the silkworm genomicDNA. Or, ranges within these ranges may be used. For example, in certainembodiments the fusion gene construct comprises a nucleic acid thatencodes for a silkworm silk gene 5′ end having a sequence as set forthin at least one of SEQ ID NO: 5, 10, 15 or 20. Also in certainembodiments, the fusion gene construct comprises a nucleic acid thatencodes for a silkworm silk gene 3′ end having a sequence as set forthin at least one of SEQ ID NO: 8, 13, 18 or 22.

In other embodiments, the present invention comprises recombinant and/orisolated DNA and/or polypeptides comprising silkworm sequences as setforth in at least one of SEQ ID NOS: 1-41, or a sequence at least 70% or75%, or 80%, or 85%, or 90%, or 95%, or 96%, or 97%, or 98% or 99%identical thereto. Thus, in certain embodiments, the present inventioncomprises an isolated DNA comprising at least one of the sequences asset forth in SEQ ID NOS: 1, 3, 5, 8, 10, 13, 15, 18, 20, 22 and 30-35.Or, the present invention may comprise an isolated DNA encoding apeptide comprising the amino acid sequence as set forth in SEQ ID NO:36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQID NO: 41. Also, in certain embodiments, the present invention comprisesrecombinant and/or isolated DNA and/or polypeptides comprising sequencesas set forth in any of the sequences disclosed herein or combinationsthereof as described herein.

The present invention comprises silkworm eggs or larvae that have beentransformed using the methods or constructs of the present invention.Thus, the silkworm eggs or larvae may comprise any of the sequencesdescribed herein.

In certain embodiments, the recombinant construct is combined with anadjuvant to increase transformation of silkworm eggs. The adjuvants maycomprise any of a number of cationic lipid preparations used fortransfection (e.g. Transfectin from Bio-Rad Laboratories, or Gene Juicefrom EMD Chemicals), but may also or alternatively include calciumphosphate preparations, or dendrimers (e.g. SuperFect from Qiagen), orhigh velocity coated nanoparticles (e.g. Gene Gun from Bio-RadLaboratories). Or, other transfection preparations and techniques knownto the art may be used.

To generate recombinant silkworms of the present invention, the methodmay comprise transforming silkworms by introducing the recombinantspider silk/silkworm silk fusion gene into a silkworm during either theegg or larval stage. Once the eggs are transformed, the method mayfurther comprise rearing the resulting larvae, and screening thesilkworms for transformation with the spider silk/silkworm silk fusiongene.

In one embodiment, the silkworm made using the methods, compositions andsystems of the present invention is capable of producing a silkcomprising a spider silk polypeptide, or an analog of a spider silkpolypeptide. In certain embodiments, the spider silk polypeptide isencoded by a recombinant DNA. In one embodiment, the spider silk gene oranalog thereof replaces a silkworm gene for silk. In one embodiment, thegene introduced into the silkworm is a fusion gene that encodes apolypeptide comprising spider silk and silkworm silk sequences (i.e., aspider silk/silkworm silk fusion). The methods may be used to introducespider silk fibroin sequences or analogs thereof into a silkworm orother producer.

In alternate embodiments, the spider silk analog may be inserted intothe silkworm heavy chain fibroin gene and/or the light chain silkwormfibroin gene. For example, in some embodiments, a spider silk chain isinserted into both the light and heavy chain loci in the silkworm. Inthis way a silk of increased strength may be produced. Or, in someembodiments, a spider silk fibroin analog is inserted into the heavychain loci in the silkworm and a different spider silk fibroin analog isinserted into the silkworm light chain loci to produce a more flexiblesilk.

In certain embodiments, recombination occurs at a single genetic locusfor fibroin. For example, recombination may occur such that a singlesilkworm light fibroin gene is excised and replaced by a spidersilk/silkworm silk fusion gene construct of the invention. Additionallyor alternatively, recombination may occur such that a single silkwormheavy fibroin gene is excised and replaced by a spider silk/silkwormsilk fusion gene construct of the invention. The present invention mayfurther comprise breeding such heterozygote to generate homozygotes ateither locus. For example, two light fibroin heterozygotes may be matedto generate offspring that are homozygous at the silkworm light chainloci for a spider silk/silkworm silk fusion gene that encodes a spidersilk fibroin, or two different spider silk fibroins, or analogs thereof.Or, two heavy fibroin heterozygotes may be mated to generate offspringthat are homozygous at the silkworm heavy chain loci for a fusion genethat encodes a spider silk fibroin, or two different spider silkfibroins, or analogs thereof.

In certain embodiments, the present invention may further comprisebreeding the recombinant silkworms to generate silkworms withrecombinant genes in both silkworm heavy loci or both silkworm lightfibroin loci by cross-breeding individuals transformed with a spidersilk/silkworm fusion gene for the silkworm light fibroin withindividuals that are transformed with a spider silk/silkworm silk fusiongene for the silkworm heavy fibroin. Or, silkworms that are heterologousat both the light and heavy chain loci (e.g., by heterologousrecombination at both the silkworm heavy and light chain genomic loci)can be mated to generate offspring that are homozygous for spider silkfusion genes for both the light and heavy fibroin loci.

Embodiments of the methods, compositions and systems of the presentinvention can thus allow for expression of spider silk analogpolypeptides in silkworms without dilution by native silkworm silkpolypeptides. Thus, in certain embodiments, at least one of the lightfibroin chain or the heavy fibroin chain in a recipient silkworm arereplaced, respectively, by a fusion gene constructs encoding a spidersilk analogs at both the light fibroin and heavy fibroin loci. Incertain embodiments, the fused gene is able to replace a native genepresent in the silkworm such that the transformed silkworm expresses apolypeptide comprising a spider silk fibroin polypeptide, or an analogthereof, and expresses significantly less of the native silkworm silk.For example, embodiments of the transformants of the present inventionmay produce not more than 80%, not more than 70%, or not more than 60%,or not more than 50%, or not more than 40%, or not more than 30%, or notmore than 20%, or not more than 10% of the native silkworm silk. In somecases, the transformants only produce silk comprising spider silkpolypeptides, or analogs thereof.

In certain embodiments of the methods, compositions and systems of thepresent invention, the recombinant DNA comprising a spider silk/silkwormsilk gene includes DNA that encodes for a non-silk polypeptide. Forexample, in certain embodiments, the recombinant DNA may includesequences that encode for a detectable protein (i.e., a reporter gene).In this way, the presence of the spider silk/silkworm silk fusion genemay be detected by monitoring the detectable protein. In one embodiment,the detectable protein may be a fluorescent protein (e.g., greenfluorescent protein) inserted downstream of the spider silk sequencesand upstream of the 3′ silkworm silk DNA sequences. Or, otherfluorescent proteins may be inserted in the same or other locations inthe polypeptide chain.

The spider silk analog peptides of the methods, compositions and systemsof the present invention may have improved characteristics as comparedto either silkworm silk or native spider silk. Thus, in certainembodiments, the methods, compositions and/or systems of the presentinvention are used to produce silkworms that express spider silk analogpeptides that are stronger, or more flexible, or more elastic, ortougher than silkworm silk. In certain embodiments, the methods,compositions and/or systems of the present invention are use to producesilkworms that express spider silk analog peptides that are stronger, ormore flexible, or more elastic or tougher than native spider silk.

For example, the spider silk analogs of the present invention mayexhibit increased strength and/or durability. In alternate embodiments,the transformed silkworm may produce a silk that is at least 10%, or15%, or 25%, or 40%, or 50%, or 75%, or 100%, or 2-fold, or 5-fold,10-fold, or 20-fold stronger than native silkworm silk. Strength isdefined as Pascals/square meter, or how much weight a given fiber cansupport.

Or, a silk having increased flexibility may be produced using themethods, compositions and systems of the present invention. In alternateembodiments, the transformed silkworm may produce a silk that is atleast 10%, or 15%, or 25%, or 40%, or 50%, or 75%, or 100% or 2-fold, or5-fold, 10-fold, or 20-fold more elastic than silkworm silk.

Additionally or alternatively, the transformed silkworm may produce asilk that is at least 10%, or 15%, or 25%, or 40%, or 50%, or 75%, or100%, or 2-fold, or 5-fold, 10-fold, or 20-fold tougher than nativesilkworm silk. As used herein, toughness is defined as the amount ofenergy that can be absorbed, or Pascals/cubic meter.

Or, the transformed silkworm may produce a silk that has at least a 10%,or 15%, or 25%, or 40%, or 50%, or 75%, or 100%, or 2-fold, or 5-fold,10-fold, or 20-fold higher breaking energy than native silkworm silk. Asused herein, breaking energy is joules/kg as opposed to toughness injoules/cubic meter.

The methods, compositions and systems of the present invention maycomprise using a single type of silkworm, or a variety of differenttypes of silkworms. In an embodiment, the silkworm comprises a silkwormof the Bombyx genus. A variety of Bombyx strains may be used including,but not limited to, Bombyx mori, Bombyx mandarina, or hybrids thereof.Or, the silkworm may comprise a silkworm of the Antheraea genus or anyother Saturniid. Also, the method may comprise transferring therecombinant fibroin genes into a plurality of varieties of silkworms bybreeding the recombinant silkworms.

For example, in one embodiment, the method may comprise a method asoutlined in FIG. 1. Thus, the method 20 may comprise the step ofligating a DNA sequence coding for a spider silk polypeptide, or ananalog thereof, between portions of the 5′ and 3′ ends of the Bombyxfibroin gene to create a fusion gene 22. The 5′ and 3′ ends of theBombyx fibroin gene should be long enough (i.e., provide sufficient DNAsequence from the native Bombyx gene) to allow for homologousrecombination to take place when the recombinant gene is placed in asilkworm host such that the native Bombyx light or heavy fibroin gene isreplaced with the corresponding light or heavy fusion (i.e., spider silkprotein/silkworm silk) gene. Either the heavy fibroin chain, the lightchain, or both, can be replaced by this procedure.

The method may also comprise the step of placing the fused gene into asuitable form for homologous recombination 24. In these embodiments, therecombinant gene may be delivered to the silkworm as a plasmid, viralvector, or naked DNA with, or without, adjuvants. The method may alsocomprise transforming silkworms by introducing the fusion gene in a formthat is suitable for homologous recombination into the silkworms eitherthe egg or larval stage 26. The method may further comprise rearing theresulting larvae and screening for transformation 28.

A variety of methods may be used to screen the eggs or larvae fortransformation. In an embodiment, a portion of the eggs or larvae areisolated and the presence of the spider silk sequences determined bydetection of the spider silk DNA (e.g., by PCR using spider silk primersand/or Southern blotting). Also, the eggs and/or larvae may be assayedfor the production of protein having the biochemical characteristics ofsilk, such as molecular weight, a silk-like appearance, and/or otherbiophysical parameters such as strength or elasticity. Or the silk orinsects can be examined for visible markers such as a fluorescentprotein included as part of the spider silk/silkworm silk construct. Forexample, measurements of physical characteristics of silk produced byrecombinant silkworms is described in the Examples herein.

The method may further comprise using standard breeding techniques togenerate silkworms homozygous for the engineered fibroin 30. Furtherbreeding can be used to generate silkworms with both the light fibroinand/or the heavy fibroin loci transformed, so as to generate homozygotesfor expression of spider silk sequences in place of either the heavy andlight chains. Additionally or alternatively, the method may comprisecross-breeding individuals having a fusion gene for a first spider silkfibroin at the light chain locus with individuals that have beentransformed with the same, or more likely, a different spider silk geneat the heavy fibroin fusion gene. The recombinant fibroin genes can alsobe moved into more desirable varieties of silkworms by standard breedingtechniques.

For example, the Bombyx mori silkworm has a single locus for the heavyfibroin gene on chromosome 25, and another single locus for the lightchain fibroin on chromosome 14 (Hyodo et al, The Japanese Journal ofGenetics, 59 (3): pp. 285-296 (1984)). There are two copies of each genein diploid cells. A transformation event would most likely occur only inone of the fibroin alleles, thus the offspring of that transformationevent would be heterozygous at that locus. Breeding such heterozygoteswould yield offspring in the ratio of 1:2:1 homozygous transformants,heterozygous transformants, homozygous wild type respectively. Testingof offspring (e.g. PCR of a few microliters of hemolymph) allows forselection of the desired homozygous individuals for further breeding.Crossing individuals homozygous for transformed heavy chain fibroin witha homozygote for transformed light chain fibroin would yield doubleheterozygotes. Crossing these offspring would yield a mix of genotypesin the ratio 1:1:2:2:4:2:2:1:1. Selection of the appropriate individualswould allow establishing a breeding population of double homozygoustransformants.

FIG. 2 shows a schematic comparison of the structural organization ofthe Bombyx mori fibroin gene (upper DNA molecule) and a synthetic spidersilk gene of the invention (lower DNA molecule). As described in moredetail herein, the spider silk analog polypeptide of the presentinvention comprises a repeated unit (denoted herein as an “internalrepeat” or I) of about 316 amino acids. The 316 amino acid internalrepeat may include 4 domains having the following sequences:LGGQGAAAAAAAAAGGGGQGG (SEQ ID NO 36), GYGGLGSQAGRGG (SEQ ID NO: 37),LGGQGAGQ (SEQ ID NO: 38), and GSGRGG (SEQ ID NO: 39) which are derivedas spider silk polypeptide consensus sequences. Or, in certainembodiments, SEQ ID NO: 38 is changed to SEQ ID NO: 41, and SEQ ID NO:36 is changed to SEQ ID NO: 40, to remove GAG regions, as describedherein.

As further described below, multiple repeats of the 316 amino acid unitmay be linked together to form a synthetic spider silk gene comprised ofbeta sheet sequences interspersed between glycine helices. The nativeBombyx silk also comprises beta sheet sequences and glycine helices, butthat the organization is different. For example, spider silks aredifferent than silkworm silk in having short beta sheets interspersedwith alpha helix or random coil sections. Thus, as compared to thespider silk analogs of the present invention, Bombyx silk hassubstantially more beta sheet domain. In other embodiments, spider silkanalogs may be designed to have proline residues so as to make themextremely elastic. Or other modifications may be made.

FIG. 3 shows a schematic representation of the method used to introducethe spider silk gene into a Bombyx silkworm. As described in more detailherein, the synthetic fusion gene is constructed to include 5′ and 3′ends that are the same sequence as the 5′ and 3′ sequence of the Bombyxfibroin gene. Thus, the synthetic fusion gene can insert itself into thelocus of the native Bombyx fibroin gene by site-specific homologousrecombination, thereby excising the fibroin gene. The excised gene isthen be degraded by nucleases.

The silkworm genes for heavy and light chain fibroin are controlled by anumber of factors. For the heavy chain there are five promoters withinthe 200 base pairs (bp) upstream of the TATA box. Also, 67 bp downstreamof the start site is an intron that appears to influence transcriptionalregulation (Takyia, F., et al., BioChem 1, 321, 645-653 (1997)). The 3′end of the heavy chain also appears to contain an essential cysteine forbonding with the light chain. The light chain has similar essentialelements in the 5′ and 3′ region (Yamaguchi, K., et al., J. Mol. Biol.210, 127-139 (1989)). By replacing the repetitive portion of the silkgenes and leaving the 5′ and 3′ regulating regions and essentialcysteine residues unchanged, optimal production of the recombinantprotein can occur. Further, by removing the native gene's repetitiveregions that encode for the majority of the silkworm silk structuralpolypeptide, there should be no production of native silkworm silk. Thisis in contrast to other systems which employ insertion of a spider silkgene at a separate location in the silkworm genome, leaving the nativesilkworm gene unaltered; in these systems, the spider silk polypeptideis expressed in combination with the silkworm silk polypeptide. In thesystem of the present invention, the spider silk peptide effectivelyreplaces the silkworm silk polypeptide.

In certain embodiments, the spider sequence is modified so as tocomprise an analog or a biological equivalent of the native sequence.There are many published sequences for protein fibers, and whichelucidate the structural properties that make for strong or very elasticfibers. Thus, the present invention includes making modifications to thestructure of the spider silk polypeptide that may enhance the functionand/or strength of the fiber.

FIG. 4A shows an embodiment of a DNA sequence (SEQ ID NO: 1) thatencodes for a spider silk polypeptide analog (SEQ ID NO: 2) which can bedenoted as an internal repeat I of the present invention. The repetitiveregion of the spider silk analog used for production of the spidersilk/silkworm silk fusion gene may be designed by comparing publishedsequences of Nephila clavipes dragline silk and determining commonlyrepeated motifs. In one embodiment, the spider silk amino acid repeatunits were combined into a 316 amino acid sequence with nine beta sheetforming regions interspersed between glycine helices, and the peptidesequences were used to derive a corresponding DNA sequence. Also, asillustrated by the embodiment shown in FIGS. 4A and 4B, the DNA sequencemay be edited to reduce repeated codons, and/or to remove selectedrestriction sites, and/or to reflect Bombyx codon biases. Also, thesequence may be modified to insert restriction endonuclease sites thatare useful for cloning. In one embodiment, a BspEI site may be added atthe 5′ end, and an XmaI site added at the 3′ end (see e.g., FIG. 4).

Spider silk undergoes a phenomenon called supercontraction. When wetted,spider silk greatly reduces its length, and becomes more plastic (Work,R. W., J. exp. Biol., 118, 379-404 (1985)). It is believed that theability of spider silk to exhibit supercontraction is due to thepresence of specific alanine residues that are encoded as GAG repeats atthe end of the polyalanine runs (Lewis, Chem. Rev., 106 (9), 3762-3774,(2006)). As increased plasticity may be an undesirable characteristicfor high strength fabrics, the GAG motifs may be edited out of the DNAsequence used to generate the synthetic fusion gene. For example, forthe sequence shown in FIG. 4A (SEQ ID NO: 2), GAG regions at the 5′ endof the beta sheet domain were converted to GAA, and GAG regions at the5′ end of the alpha helix domain were converted to GGG by modifying thenative sequence domains (SEQ ID NO: 3) to generate the alternate spidersilk peptide (SEQ ID NO: 4) as shown in FIG. 4B.

In certain embodiments, the spider silk polypeptide, or analog thereofmay comprise a single spider silk internal repeat domain (I) ofalternating beta sheet domains (dark regions) alternating with alphahelix domains (light colored regions) such as IA, IB, IC, or ID shown inFIG. 4C. Thus, the beta sheet regions, alpha helix regions and/or numberof each may be varied to produce different internal repeats that canencode a spider silk polypeptide or analog thereof. Or, the spider silkpolypeptide or analog thereof may comprise a plurality of the same ordifferent single spider silk internal repeat domains (e.g., IA, IB, IC,ID and the like) linked end to end in various arrangements as depictedin FIG. 4C for constructs (i)-(vi).

In one embodiment, the 5′ genomic sequence from the silkworm gene startsat the ATG start codon, and ends 33 nucleotides after the end of thefirst intron. Or, shorter or longer 5′ regions may be used. In certainembodiments, however, the 5′ non-repetitive silkworm sequence is longenough such that homologous recombination with the 5′ end of the nativesilkworm gene can occur. Embodiments of 5′ heavy chain DNA sequences forvarious species of silkworm are shown as SEQ ID NOS: 5, 10 and 15 inFIGS. 5, 7 and 9, respectively. Also, an embodiment of a 5′ light chainfibroin sequence is shown as SEQ ID NO: 20 (FIG. 11). The 5′ heavy chainfibroin sequences as shown as SEQ ID NO: 5, 10, and 15 have the upstreampromoters intact, and the intron in its normal position. Thus, thesesequences (promoter and intron) will still be in the correct position inthe silkworm genome after homologous recombination.

In one embodiment, the sequence used as the 5′ sequence may be modifiedto include a restriction endonuclease site at the 3′ end of thesequence. In an embodiment, the restriction endonuclease site matchesthe restriction endonuclease site at the 5′ end of the internal repeatsequences. For example, in one embodiment, the 5′ heavy or light chainsequence comprises a BspEI restriction site at the 3′end to match withthe internal repeat sequence 5′ restriction site (SEQ ID NOS: 5, 10, 15and 20).

As described herein, the fusion gene construct may also include asequence to promote homologous recombination of the 3′ end of theconstruct with the native silkworm gene. An example of 3′ sequences fromvarious silkworm species that may be used is shown as SEQ ID NOS: 8, 13,and 18 in FIGS. 6, 8, and 10, respectively. Also, an embodiment of a 3′light chain fibroin sequence is shown as SEQ ID NO: 22 (FIG. 12). In anembodiment, the 3′ sequence may start 57 nucleotides upstream of thecritical cysteine residue found in the native silkworm gene, andcontinue for about 664 nucleotides downstream of the terminal cysteine.The sequence may also comprise an in-frame XmaI site at the 5′ end tomatch a 3′ restriction site present on the internal repeat sequence(i.e., SEQ ID NOS: 8, 13, 18 and 22).

In an embodiment, the gene sections may be inserted into a vector. Incertain embodiments, the sections are combined in a manner such thatmultiple repeats of the spider silk polypeptide (e.g., multiple repeatsof SEQ ID NO: 1) are linked end to end. For example, the gene sectionsmay be inserted into pUCminus (pUC⁻) plasmids (Blue Heron Bio). Theassembly of the synthetic fusion gene may then employ a series ofligation reactions as outlined in FIG. 13. First, the plasmidscontaining the 5′ section (p5′) and the internal repeating section (pI)may each be double digested with NcoI and BspEI. The resulting DNAfragments may then be gel purified, and the 5′ DNA sequence ligated withthe digested pI′ to produce plasmid p5I′ (FIG. 13).

Next, a plasmid with the 3′ section (i.e., p3′) may be digested withXmaI and NcoI, and plasmid P5′I may be digested with BspEI and NcoI.After gel purification, the digested p3′ and the 5I segment may beligated to form plasmid p5′I3′ (FIG. 13). Purified p5′I3′ may then besplit into two fractions and digested with NcoI/BspEI and NcoI/XmaI. TheNcoI/BspEI digested plasmid contains the 3′I section of the gene with asticky end recognized by an XmaI digest; the digested plasmid maytherefore be ligated with the purified XmaI/NcoI digested section togive a plasmid with the 5′ section, two internal repeats, and the 3′section (p5′I²3′) (FIG. 13). This method of joining the sectionsdestroys the restriction site between the repeating sections. Repeatingthe dual double digests and ligation allows the repeats to be generatedin any number from 1 to as large as the vector and recombinase-minushost are able handle, or p5′I^(x)3′ with x being any number from 1 tothe limit of the vector/host. To produce repeat numbers outside of theseries 1, 2, 4, 8, 16, 32 and so forth, would require using two plasmidhaving the required number of repeats such that the sum of the repeatsprovides the desired end-product. For example, to produce p5′I³3′,plasmid p5′I²3′ (containing 2 insert repeats) could be digested withBspEI/NcoI, and p5′I3′ (containing 1 insert repeat) digested withXmaI/NcoI. Alternatively and/or additionally, different plasmids havingdifferent internal repeats (e.g., such as are depicted as IA, IB, IC,and ID of FIG. 4C) may be used to generate concatamers of differentrepeat units ligated together (e.g., p5′IA₄IB₅₀IC₂3′ and the like).Ligation of the appropriate DNA pieces would produce the desiredplasmid. This method of concantenation using a combination ofisoschizomers and a common restriction site is broadly applicable toassembling repetitive gene constructs.

After construction of the gene, injection into either eggs or larvae canresult in homologous recombination in some of the individuals. These canbe identified with PCR screening, and used to establish colonies oftransformed silkworms. Methods of injection and adjuvants that encourageDNA uptake and transformation/recombination are known in the art, as aremethods of raising the silkworms and using PCR to screen for the newgenes.

Also, there are abundant marker genes that can be used to identify atransformation event. Many fluorescent proteins are readily available inplasmid form, and can be inserted in-frame into the silk sequence. Forexample to insert a GFP gene in-frame, a plasmid can be created with aGFP gene flanked 3′ with a BspEI site and a combined XmaI/NcoI site onthe 5′ end. This can then be used in a scheme similar to that in FIG. 14to produce a construct with multiple repeats (e.g., the same I, ordifferent I, such as IA, IB, IC or ID) linked to a reporter gene. Thesilk gland in the larvae and the spun silk can then fluoresce greenunder appropriate illumination. Alternatively, the fluorescent proteincan be paired with a promoter and inserted into the intron located atthe 5′ end of Saturniid fibroin genes. As an example, a GFP gene using asericin promoter and 3′ polyadenylation sequence is in FIG. 15. Use ofBglII and XhoI restriction enzymes will allow the insertion of thisconstruct into the intron of Bombyx mori fibroin, and result in GFPexpression in the sericin layer of silk. This will be visible in thelarvae and the silk. It will also be removable from the spun silk duringthe washing process, unlike the in-frame insertion.

The same process used to replace native fibroin genes with a new silkgene can also be used to knockout expression of the native gene.Homologous recombination that removes promoters, start sequences, amajor portion of the gene, or introduces stop codons, will result innon-production of silk from that location on the chromosome. If othertransformation processes such as random insertion using baculovirusand/or transposase mediated insertion are used to insert functional silkgenes elsewhere in the genome, these would be produced without dilutionby native silk molecules. The 5′ portion of a knockout homologousrecombination gene for the heavy chain fibroin is shown in FIG. 16.

EXAMPLES

The invention will be illustrated in further detail with the followingexamples.

Example 1—Design of the 5′ and 3′ Homologous Ends

The NCBI Entrez database was used to determine the sequence of theBombyx mori heavy chain fibroin gene. Wu and Cao (J Zheijian Univ Sci.,(2004) 5(6):644-650) had success using homologous sections of about 1kb, Rubnitz and Subramani (Mol Cell Biol. 1984 November; 4(11):2253-2258) report a sharp drop in efficiency below 214 bp. The 5′homologous section contains an intron of about 1 kb, and ˜30 bp wasadded to each end to avoid possible effects on post-translationalprocessing. To facilitate possible further use of the gene constructwith other promoters, organisms, etc., the decision was made to beginthe 5′ sequence with the ATG start codon. This leaves the upstreampromoters untouched and the intron in its normal place after homologousrecombination. The 5′ sequence was modified to have a BspEI restrictionsite at the 3′ end to match with the internal repeat sequence 5′restriction site (FIG. 5).

The 3′ sequence was chosen to start 57 nucleotides upstream of thecritical cysteine residue, and continued 661 nucleotides downstream ofthe stop codon. The 3′ sequence has an in-frame XmaI site at the 5′ endto match the internal repeat sequence 3′ restriction site (FIG. 6).Thus, both the 5′ sequence and the 3′ sequence include the essentialelements while coding for a minimal amount of the peptide sequence. Thegene constructs were ordered from Blue Heron Biotechnology, (Bothel W A98021 USA) inserted into their pUC Minus plasmid.

Example 2—Design of the Internal Repeat Segment

NCBI Entrez was consulted to obtain published sequences of Nephiladragline silk which were used to determine commonly repeated motifs.These were reduced to 13 commonly occurring sequences, 4 of which areheavily represented in the highly repetitive portion of the gene:LGGQGAGAAAAAAAGGAGQGG (SEQ ID NO: 40), GYGGLGSQAGRGG (SEQ ID NO: 37),LGGQGAGQ (SEQ ID NO: 38), and GSGRGG (SEQ ID NO: 39). The sequences (SEQID NOS: 37, 38, and 39) were arranged into higher order motifs toreflect an approximation of the natural gene. The 316 amino acidsequence with nine beta sheet forming regions (shown in FIG. 4A) wastranslated into a DNA sequence and edited to reduce repeated codons,remove selected restriction sites, and reflect Bombyx codon biases. ABspEI site was added at the 5′ end, and an XmaI site at the 3′ end.

Spider silk undergoes a phenomenon called supercontraction. When wetted,it greatly reduces its length, and becomes more plastic Work, R. W., J.exp. Biol., 118, 379-404 (1985). Lewis, Chem. Rev., 106 (9), 3762-3774,(2006), reports this is probably due to specific alanine residues, foundin GAG repeats at the end of the polyalanine runs. As increasedplasticity when wet can be an undesirable characteristic for highstrength fabrics, the GAG motifs were edited out by single base changes.In general, 5′ GAG repeats found in the spider silk beta sheet domain(see e.g., SEQ ID NO: 40) were converted to GAA, and 3′ GAG wereconverted to GGG as shown in FIG. 4B (see e.g., SEQ ID NO: 36). Also, asimilar change can be made to SEQ ID NO: 38 to generate SEQ ID NO: 41for use in spider silk internal repeat constructs. The DNA sequence usedas the spider silk repeat unit is shown as FIG. 4A (SEQ ID NO: 1).

Example 3—Assembling the Gene

The three gene segments had NcoI, BspEI, or XmaI restriction sites addedso that they would remain in-frame when joined end to end, i.e., the5′end (SEQ ID NO: 5) joined to the spider silk internal repeat (I) (SEQID NO: 1) joined to the 3′ sequence (SEQ ID NO: 8). Plasmid p5′containing the Bombyx 5′ homologous end (5′ end, or 5′) (SEQ ID NO: 5)and plasmid pI containing the internal repetitive section (I) (SEQ IDNO: 1) were digested with BspEI and NcoI. The digests were then run onagarose gel. The cut pI and 5′ end bands were extracted with a glassmilkprocedure and 0.1 μg of each were ligated and transformed into NEB's10-beta E. coli to produce plasmid p5′I. Plasmids p5′I and p3′(containing the Bombyx 3′ homologous end) were digested with XmaI andNcoI. The 5′I section and the cut p3′ plasmid were gel purified and 0.1μg of each were ligated and transformed into 10-beta E. coli, togenerate plasmid p5′I3′. A preparation of p5′I3′ was split and halfdigested with XmaI/NcoI, and the other half digested with BspEI/NcoI.The 5′I section produced by the XmaI/NcoI digest and the cut pI3′ wereagain gel purified, ligated, and transformed to produce p5′I²3′ (shownas SEQ ID NO: 32 in FIG. 19).

As indicated in FIG. 13, the process of dual double digests and ligatingcan continue to produce a plasmid with the 5′ and 3′ ends and any numberof insert repeats. To produce repeat numbers outside of the series 1, 2,4, 8, 16, 32 and so forth, would require using two plasmid having therequired number of repeats such that the sum of the repeats provides thedesired end-product. For example, to produce p5′I³3′, plasmid p5′I²3′(containing 2 insert repeats) could be digested with BspEI/NcoI, andp5′I3′ (containing 1 insert repeat) digested with XmaI/NcoI. Ligation ofthe appropriate DNA pieces would produce the desired plasmid.

Example 4—Transformation of Silkworms with p5′I²3′

p5′I²3′ was prepared using Novagen's Insect Gene Juice as per themanufacturer's directions, using Bio-Whitaker's Insect X-press serumfree media for dilution. One hundred late third instar larvae wereinjected with 25 μl of transfection mix; about 300 ng plasmid perinsect. A Hamilton 50 μl syringe with a 30 gauge #4 point needle wasinserted laterally near the 5^(th) abdominal segment, run up to near the4^(th) abdominal segment, and the media injected slowly. The needle waskept in for about 5 seconds after the injection was complete to allowfor equilibration: too early a removal resulted in some of the mediabeing expelled.

The injected larvae were placed in a 27° C. incubator at ˜70% humidityand fed on artificial diet until pupation. There was a 98% survival rateduring the two weeks to pupation, but 20% died during the wandering orspinning stages, and 30% of the survivors failed to emerge on their own.The surviving enclosed adults were paired on starch paper and allowed tomate. After two hours the males were separated and the females allowedto lay eggs. Twenty-two egg masses resulted and were given sequentialbatch numbers.

Four days after laying, the egg masses had approximately 25% of theireggs removed and the genomic DNA extracted with a cartridge based kit.PCR using primers specific to the inserted repetitive region or thenative sequence revealed two strong transformation events, that is, apositive PCR for the transgene and a weak or absent response to thegenomic primers. Three others indicated a more balanced mixture oftransgenic and native sequence, and four others showed anomalousbanding, not clearly one or the other. All eggs masses were placed in a4° C. chamber to complete diapause.

After one month four egg masses were selected and brought to roomtemperature for three hours. They were then soaked in 4.5 M HCl at 48°C. for five minutes, washed three times with water and allowed toair-dry. The eggs were placed in a humid 27° C. incubator. At 10-12 daysthe eggs began to hatch, and emergence over three days was essentially100%. The larvae were fed on artificial diet and placed in pupationcontainers when they reached the wandering stage. After one week thecocoons were carefully removed, the floss gently stripped off, and thestrength and elasticity measured with a tensiometer. This wasaccomplished by carefully stripping single fibers of 20-30 cm from thecocoon. These were fastened to the tensiometer and a ten cm sectionstrained at approximately 3%/sec. Five to ten segments were tested fromeach cocoon. Silk from untreated larvae was used as the control. Thecocoons were placed in labeled individual containers in the incubatorand left to emerge.

Results

The transformed silk was significantly different from natural silk. Thestrength of natural silk was measured at 0.78±0.06 GPa. The transformedsilk showed two curves (FIG. 23), with means of 1.02±0.04 GPa and1.095±0.03 GPa. Elasticity was also significantly different. Naturalsilk had an elasticity of 14±1.5%, while the transformed silk could bedivided into two groups, high and low elasticity, measuring 22±0.7% and33±1.0%, respectively (Table 1). It can be seen that for both types oftransformants (high and low elasticity) the strength was about the same,and significantly greater than the native silk.

Table 2 shows a comparison of the strength, elasticity, toughness andbreaking energy for wild-type silkworm silk (i.e., Bombyx silk), and thesilk of silkworms transformed with two different constructs of theinvention: the 5′-I²-3′ construct (SEQ ID NO: 32) (Trans/Silk 2 rpt) andthe 5′-I²-3′ construct (SEQ ID NO: 34) (Trans/Silk 3 rpt), as comparedto literature values for spider silk, silkworm silk (Bombyx silk), andKEVLAR™. The transformed silk compares very closely with natural spidersilk in toughness and elasticity. The two ranges of strength andelasticity for the transformed silk seems to indicate that mating of theG0 transformed insects resulted in a number of homozygous transformedindividuals in the G1 generation; about 21%, or close to the expectedMendelian result for two heterozygous parents. This result may be due toselecting egg masses that showed strong PCR evidence of transformationfor rearing.

TABLE 1 Elasticity (%) SD* 95% Confidence Natural Silk 14.059 3.85 ±1.54Low Transformed 21.562 4.00 ±0.69 High Transformed 32.718 2.86 ±0.91Strength (GPa) SD 95% Confidence Natural Silk 0.776 0.142 ±0.60 LowTransformed 1.016 0.219 ±0.04 High Transformed 1.095 0.159 ±0.03*Standard Deviation

TABLE 2 Strength Elasticity Toughness Breaking Energy Literature Pascals% Joule/cubic M Joules/kg Spider Silk 1.10E+09 35 1.60E+08 4.00E+05Bombyx Silk 6.00E+08 18 7.00E+07 3.00E+04 Kelvar 3.60E+09  3 5.00E+073.30E+04 Measured Pascals % Joule/cubic M Joules/kg Trans. Silk 2 rpt1.09E+09 33 1.90E+08 3.85E+05 Trans. Silk 3 rpt 1.86E+09 36 2.68E+085.31E+05 Bombyx Silk 7.99E+08 14 8.40E+07 1.68E+05

Also, FIG. 24 shows a schematic representation of the results of a PCRof DNA extracts from a portion of some of the second generation eggmasses transformed with a pUC-plasmid containing a 5′4²-3′ insert (i.e.,SEQ ID NO: 32). In these experiments, the DNA from about 75 secondgeneration eggs (25% of the total) from individual matings (i.e., eachsample corresponds to a different male-female mating) was pooled and PCRconducted using a primer internal to the 3′ non-repetitive section ofthe native silkworm heavy fibroin gene, and either a primer specific toeither the synthetic spider silk derived internal repetitive region(i.e., spider silk analog) (Panel A) or a primer specific to the nativesilkworm heavy fibroin gene (Panel B). The PCR products from randomsamples (14-18, and 20) were compared to PCR products generated with DNAcorresponding to the plasmid with the silkworm/spider silk gene (P) andBombyx mori genomic DNA (G). It can be seen that samples 18 and 20 showa pattern distinct from both genomic DNA and plasmid DNA indicatingsuccessful integration of the silkworm silk/spider silk construct intothe silkworm genome.

Example 5—Other Silk Producing Moths

The same process can be used in any silk producing Lepidoptera. Bombyxmandarina, the wild form of the common silkworm, has essentiallyidentical 5′ and 3′ regions on the fibroin genes, and can be transformedusing the same reagents constructed to transform Bombyx mori. The otherSaturniids are also capable of being transformed using this technique,in particular the species in the genera Antherae which are also used forsilk production. For example to transform Antheraea peryni, the ChineseOak Silkmoth, the 5′ and 3′ homologous end segments could be changed tothe sequences in FIGS. 7 and 8. To transform Antheraea yamamai, theJapanese Oak Silkmoth, the sequences in FIGS. 9 and 10 may be used.Similarly, the sequences for light chain fibroin may be inserted into avector comprising silkworm 5′ and 3′ sequences (e.g., SEQ ID NOS: 20 and22) shown as FIGS. 11 and 12. The internal repeat sequences and geneassembly could be the same as the process described above.

The general technique for transforming the Saturniids is to obtain thesequence of the fibroin, typically through cDNA sequencing, and useapproximately 500 (range 200-2000) or more base pairs from the 5′ and 3′end along with an internal repeat segment to construct the artificialfibroin gene used for homologous recombination.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to certain embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1.-22. (canceled)
 23. A method of making a transgenic silkworm thatproduces a silk comprising a spider silk polypeptide, or an analog of aspider silk polypeptide: a) providing a silkworm, wherein the silkwormis Bombyx mori, Bombyx mandarina, Antheraea peryni, or Antheraeayamamai; b) transforming the silkworm of step (a) by inserting a nucleicacid sequence encoding a spider silk fibroin polypeptide, or an analogof a spider silk fibroin polypeptide, into the genome of the silkworm,wherein the nucleic acid comprises at least 2800 nucleotides encodingalternating beta-sheet domains and alpha-helix domains, wherein theamino acid sequence of the beta-sheet domains are each at least 60%identical to at least one of the beta-sheet domains as set forth in SEQID NO: 2, and wherein expression of the native silkworm fibroin gene isreduced; and c) selecting for a transgenic silkworm from step (b) thatproduces a silk that is at least 50% stronger than native silkworm silk.24. The method of claim 23, wherein the silkworm is Bombyx mori orBombyx mandarina.
 25. The method of claim 23, wherein the silkworm isAntheraea peryni or Antheraea yamamai.
 26. The method of claim 23,wherein the analog of the spider silk fibroin polypeptide comprisesabout 30 to 1000 spider silk beta-sheet domains alternating with about30 to 1000 spider silk alpha helix domains.
 27. The method of claim 23,wherein the nucleic acid sequence encoding the analog of the spider silkfibroin polypeptide encodes a polypeptide comprising the amino acidsequence as set forth in at least one of SEQ ID NO: 36, SEQ ID NO: 37,SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or SEQ ID NO:
 41. 28. Themethod of claim 23, wherein the nucleic acid sequence encoding theanalog of the spider silk fibroin polypeptide comprises the amino acidsequence as set forth in SEQ ID NO:
 2. 29. The method of claim 23,wherein the nucleic acid sequence encoding the analog of the spider silkfibroin polypeptide further comprises a reporter gene.
 30. The method ofclaim 23, wherein the transgenic silkworm produces a silk that is atleast 40% more elastic than native silkworm silk.
 31. The method ofclaim 23, wherein the method is used to produce a first population oftransformed silkworms by transforming a first set of silkworms with afirst nucleic acid sequence encoding a first spider silk fibroinpolypeptide, or an analog of the first spider silk fibroin polypeptide,and wherein the method is used to produce a second population ofsilkworms by transforming a second set of silkworms with a secondnucleic acid sequence encoding a second spider silk fibroin polypeptide,or an analog of the second spider silk fibroin polypeptide.
 32. Themethod of claim 31, further comprising breeding at least one of thetransformed silkworms from each of the first and second populations togenerate silkworms heterozygous for the first and second spider silkpolypeptides and/or analogs thereof.
 33. An isolated nucleic acid thatencodes a spider silk fibroin polypeptide or an analog of a spider silkfibroin polypeptide, operably linked to at least one silkworm fibroinnucleic acid sequence, wherein the silkworm is Bombyx mori, Bombyxmandarina, Antheraea peryni, or Antheraea yamamai.
 34. The method ofclaim 33, wherein the silkworm is Bombyx mori or Bombyx mandarina. 35.The method of claim 33, wherein the silkworm is Antheraea peryni orAntheraea yamamai.
 36. The isolated nucleic acid of claim 33, whereinthe at least one silkworm nucleic acid sequence comprises a 5′ end of anative silkworm fibroin gene, a 3′ end of a native silkworm fibroingene, or both a 5′ end of a native silkworm fibroin gene and a 3′ end ofa native silkworm fibroin gene.
 37. The isolated nucleic acid of claim36, wherein the analog of the spider silk fibroin polypeptide is encodedby a nucleic acid sequence comprising at least 2800 nucleotides encodingalternating beta-sheet domains and alpha-helix domains, wherein theamino acid sequence of the beta-sheet domains are each at least 60%identical to at least one of the beta-sheet domains as set forth in SEQID NO: 2, and wherein the analog of the spider silk fibroin polypeptideis at least 50% stronger than native silkworm silk.
 38. The isolatednucleic acid of claim 36, wherein the silkworm fibroin gene 5′ endcomprises a nucleic acid sequence as set forth in at least one of SEQ IDNOs: 5, 10, 15, or
 20. 39. The isolated nucleic acid of claim 36,wherein the silkworm fibroin gene 3′ end comprises a nucleic acidsequence as set forth in at least one of SEQ ID NOs: 8, 13, 18, or 22.40. A transgenic silkworm transformed with an isolated nucleic acid thatencodes a spider silk fibroin polypeptide or an analog of a spider silkfibroin polypeptide, wherein the analog of the spider silk fibroinpolypeptide is encoded by at least 2800 nucleotides encoding alternatingbeta-sheet domains and alpha-helix domains, wherein the amino acidsequence of the beta-sheet domains are each at least 60% identical to atleast one of the beta-sheet domains as set forth in SEQ ID NO: 2,wherein the transgenic silkworm produces a silk that is at least 50%stronger than native silkworm silk, and wherein the silkworm is Bombyxmori, Bombyx mandarina, Antheraea peryni, or Antheraea yamamai.
 41. Asilk produced by the transgenic silkworm of claim 40, wherein the silkis at least 50% stronger than native silkworm silk.